The objective of this project is to develop a novel protein expression and purification system which has the advantages of the GST or MBP systems without the drawbacks of additional proteolytic cleavage steps. To achieve this, protein splicing elements (named inteins) are utilized for their ability to catalyze highly specific peptide bond cleavage reactions. The design of the system is as follows: a target protein is fused to the C-terminus of a modified intein in which the chitin-binding domain is inserted to allow affinity purification and an MBP fragment is fused to the N-terminus of the intein to allow favorable translational start. The focus of the project is to investigate induction of peptide bond cleavage at the C-termini of the intein by temperature, pH and/or a thiol reagent. All mini-inteins (those that lack the endonuclease domain) will be tested for their ability to improve protein expression and allow secretion of the fusion protein in yeast. Moving from shake-flask to a small fermentor, the investigation will also examine control of cleavage and secretion under high-cell-density fermentation conditions. The goal is to make this intein-mediated purification system immediately applicable to large-scale commercial production of recombinant proteins. PROPOSED COMMERCIAL APPLICATIONS: This research leads to the development of a novel protein expression and purification system which may significantly simplify the purification process of recombinant proteins and may be immediately applicable to large-scale commercial production of therapeutic proteins.